Population studies, direct-to-consumer genetic tests, biobanks, and other large-scale genomic research projects rely on DNA gathered from saliva samples, which are often self-collected and submitted by the general public. Unlike blood, saliva samples remain stable at room temperature for years, making them an ideal sample type for large or long-term studies. However, concerns about the quality and amount of DNA that can be extracted from saliva have so far limited its use in long-read sequencing, where high-quality, high-molecular-weight (HMW) DNA is key to performance.
To overcome this hurdle, researchers at the Garvan Institute of Medical Research in Australia used Oragene™ collection kits (DNA Genotek™) and the Nanobind® extraction kits (PacBio) to sequence saliva samples collected several years ago in remote communities.
This proof of principle study demonstrates that when blood samples are not available, saliva can be a viable alternative for obtaining high-quality long-read sequencing data.
| Saliva samples | Blood samples |
|---|---|
| Collection is noninvasive, can be done by non-experts and in remote locations | Collection requires a needle, a trained technician, and a sterile laboratory setting |
| Stable at room temperature for years | Must be refrigerated or frozen immediately after collection |
| Cost-effective shipping and storage | Must be shipped on cold packs |
Nanobind extraction methods yield high-molecular-weight DNA
Individuals from the communities in this study provided their own saliva samples using Oragene™ self-collection kits (pictured below). Dr. Ira Deveson’s team at the Garvan Institute then extracted HMW DNA from 500 µL of saliva using the Nanobind CBB kit.
First, Nanobind kits process and lysate cells using reagents specific to the sample type. Next, the DNA binds to magnetic disks, which shield the DNA from damage during the extraction process. Finally, a size selection step uses a short-read eliminator (SRE) kit to remove DNA fragments below 10 kb. This three-step process results in HMW DNA that is ready for HiFi long-read sequencing.
In this study, DNA was sheared to a size of 15-20 kb. Libraries were prepared and sequenced on a single SMRT Cell on the Revio long-read system. The researchers then performed variant calling and de novo assembly using the resulting HiFi sequencing reads.
For more about extracting HMW DNA from saliva samples using the Oragene™ device and Nanobind kits, see the extraction protocol and tech note.
Results “exceed expectations” for challenging saliva samples
Using the Nanobind kits, Dr. Deveson and team recovered 1–45 µg of pure HMW DNA from the saliva samples. This HMW DNA was optimal for HiFi sequencing— the research team obtained 60 to 114 Gb of HiFi sequencing data on the Revio system. That translated to ~20x to ~40x coverage per genome, which enabled de novo assembly and comprehensive variant detection across the genome.
“These numbers exceeded my expectations, given the challenging nature of these saliva samples, which were collected several years ago in remote Indigenous communities in Northern Australia.”
—Dr. Ira Deveson, Garvan Institute of Medical Research
With the right protocol, saliva is a viable DNA source for long-read sequencing
This proof of principle study demonstrates that HiFi sequencing can successfully sequence DNA from saliva samples, even those that have stood the test of time. Getting high molecular weight DNA from saliva is now possible, using Oragene™ collection kits and the Nanobind extraction protocol.
The authors conclude that because saliva samples offer numerous advantages over traditional blood samples and buccal swabs, using this approach has the potential to aid in recruitment and make population studies easier both for researchers and the citizen scientists involved.
“When blood is not available, saliva can be a viable alternative for obtaining high-quality long-read sequencing data.”
—Dr. Ira Deveson, Garvan Institute of Medical Research
Get started with PacBio
For details about using DNA from saliva samples in your own long-read sequencing projects research, see the DNA extraction protocol and tech note.
Nanobind CBB and PanDNA kits are designed for human or animal blood, human saliva, mammalian cultured cells, human or animal tissues, and bacteria. Learn more about Nanobind technology or speak to a PacBio scientist about getting started with the Revio long-read sequencer.